Cytomegalovirus Pathogenesis Immunodeficiency Human cytomegalovirus (CMV) is a major pathogen affecting the developing fetus and immunocompromised individuals. This virus is an important opportunistic pathogen and possible cofactor in AIDS. This grant will dissect the regulatory role of three murine CMV genetic elements, the enhancer, an alpha gene (ie2) and a beta gene (sgg1) in latency, pathogenesis and tissue tropism. First, the function of ie2 and the enhancer in latent infection will be assessed using viral mutants. Work will focus on these two genetic elements because they are already known to influence latency, possibly through transcriptional regulation. Preliminary work has established that ie2 is important for efficient reactivation of latent murine CMV and that the human CMV enhancer can complement the ie2 defect in cis. Further, ie2 has significant sequence similarity to the Epstein-Barr virus Z transactivator, which is thought to regulate reactivation. The functional domains of ie2 will be identified through construction of viral mutants, particularly in putative leucine zipper and charged domains of ie2. The function of ie2 as a transactivator of the enhancer will be investigated using transient transfection assays and the importance of the enhancer and enhancer elements as targets for ie2-mediated transactivation will be determined using viral mutants and transient assays. Finally, the human CMV enhancer elements that confer efficient reactivation on ie2_ murine CMV mutants will be mapped. Second, the regulatory role of the enhancer, ie2 and sgg1 in tissue tropism and pathogenesis will be investigated. Growth and pathogenesis will be evaluated in normal, immunocompetent BALB/c and C3H mice, to reveal the influence of host genetic factors, as well as in immunodeficient, SCID mice (BALB/c without functional T and B cells) to reveal the importance of a functional cellular immune system in observed phenotypic differences. Although it is well established that murine CMV pathogenesis differs significantly in regards to age, genetic background and immune status, little is known about the contribution of specific viral genetic element to this process. Viral plaque assays as well as sensitive in situ assays for the detection, quantitation and localization of recombinant virus and viral expression in infected tissues will be used to help determine the phenotype of different mutants. We will use the expression of beta-galactosidase from wild type and mutant viruses to study viral gene expression in cells that harbor acute, persistent and latent virus, paying particular attention to differences between wild type and mutant viruses. This analysis will identify the cell type(s) in which murine CMV remains latent. This project will provide direct information on specific mechanisms of murine CMV latency, tissue tropism and pathogenesis. Based on the presumed or established existence of genetic elements similar to the enhancer, ie2 and sgg1 in human CMV, an attempt will be made to test function of human CMV homologues in the murine CMV genome. Should this be possible, specific information on the role of human CMV genetic elements in latency or pathogenesis may result from this grant as well.